Drug therapy for celiac sprue

ABSTRACT

A ministering an effective dose of a tTGase inhibitor to a Celiac or dermatitis herpetiformis patient reduces the toxic effects of toxic gluten oligopeptides, thereby attenuating or eliminating the damaging effects of gluten.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/380,761 filed May 14, 2002; to U.S. Provisional Application60/392,782 filed Jun. 28, 2002; and to U.S. Provisional application No.60/422,933, filed Oct. 31, 2002, and to U.S. Provisional Application60/428,033, filed Nov. 20, 2002, each of which are herein specificallyincorporated by reference.

BACKGROUND OF THE INVENTION

In 1953, it was first recognized that ingestion of gluten, a commondietary protein present in wheat, barley and rye causes a disease calledCeliac Sprue in sensitive individuals. Gluten is a complex mixture ofglutamine- and proline-rich glutenin and prolamine molecules and isthought to be responsible for induction of Celiac Sprue. Ingestion ofsuch proteins by sensitive individuals produces flattening of thenormally luxurious, rug-like, epithelial lining of the small intestineknown to be responsible for efficient and extensive terminal digestionof peptides and other nutrients. Other clinical symptoms of Celiac Sprueinclude fatigue, chronic diarrhea, malabsorption of nutrients, weightloss, abdominal distension, anemia, as well as a substantially enhancedrisk for the development of osteoporosis and intestinal malignanciessuch as lymphoma and carcinoma. The disease has an incidence ofapproximately 1 in 200 in European populations and is believed to besignificantly under diagnosed in other populations.

A related disease is dermatitis herpetiformis, which is a chroniceruption of the skin characterized by clusters of intensely pruriticvesicles, papules, and urticaria-like lesions. IgA deposits occur inalmost all normal-appearing and perilesional skin. Asymptomaticgluten-sensitive enteropathy is found in 75 to 90% of patients and insome of their relatives. Onset is usually gradual. Itching and burningare severe, and scratching often obscures the primary lesions witheczematization of nearby skin, leading to an erroneous diagnosis ofeczema. Strict adherence to a gluten-free diet for prolonged periods maycontrol the disease in some patients, obviating or reducing therequirement for drug therapy. Dapsone, sulfapyridine, and colchicinesare sometimes prescribed for relief of itching.

Celiac Sprue (CS) is generally considered to be an autoimmune diseaseand the antibodies found in the serum of the patients support the theorythat the disease is immunological in nature. Antibodies to tissuetransglutaminase (tTGase or tTG) and gliadin appear in almost 100% ofthe patients with active CS, and the presence of such antibodies,particularly of the IgA class, has been used in diagnosis of thedisease.

The large majority of patients express the HLA-DQ2 [DQ(a1*0501, b1*02)]and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villous atrophy of the small intestine.

At the present time, there is no good therapy for the disease, except toavoid completely all foods containing gluten. Although gluten withdrawalhas transformed the prognosis for children and substantially improved itfor adults, some people still die of the disease, mainly adults who hadsevere disease at the outset. A leading cause of death islymphoreticular disease, especially intestinal lymphoma. It is not knownwhether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

Gluten is so widely used, for example, in commercial soups, sauces, icecreams, hot dogs, and other foodstuffs, that patients need detailedlists of foodstuffs to avoid and expert advice from a dietitian familiarwith celiac disease. Ingesting even small amounts of gluten may preventremission or induce relapse. Supplementary vitamins, minerals, andhematinics may also be required, depending on deficiency. A few patientsrespond poorly or not at all to gluten withdrawal, either because thediagnosis is incorrect or because the disease is refractory. In thelatter case, oral corticosteroids (e.g., prednisone 10 to 20 mg bid) mayinduce response.

In view of the serious and widespread nature of Celiac Sprue and thedifficulty of removing gluten from the diet, better methods of treatmentare of great interest. In particular, there is a need for treatmentmethods that allow the Celiac Sprue individual to eat gluten-containingfoodstuffs without ill effect or at least to tolerate such foodstuffs insmall or moderate quantities without inducing relapse. The presentinvention meets this need for better therapies for Celiac Sprue byproviding new drugs and methods and formulations of new and existingdrugs to treat Celiac Sprue. International Patent ApplicationUS03/04743, herein specifically incorporated by reference, disclosesaspects of gluten protease stability and immunogenicity.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for treatingCeliac Sprue and/or dermatitis herpetiformis and the symptoms thereof byadministration of a tTGase (tissue transglutaminase) inhibitor to thepatient. In one embodiment, the tTGase inhibitor employed in the methodis a known small molecule-tTGase inhibitor selected from the groupconsisting of vinylogous amides, sulfonamides,2-[(2-oxoalkyl)thio]imidazolium compounds, diazoketones, and3-halo-4,5-dihydroisoxazoles. In another embodiment, the tTGaseinhibitor is a dipeptide mimetic, a compound that mimics in structure adipeptide selected from the group consisting of PQ, PY, QL, and QP.

In another aspect, the present invention provides novel tTGaseinhibitors and methods for treating Celiac Sprue and/or dermatitisherpetiformis by administering those compounds. In one embodiment, thetTGase inhibitor is a peptide or peptidomimetic that has or containswithin a longer sequence the structure of the peptide PQPQLPY or PQPELPYin which the E or the second Q is replaced by a glutamine mimetic thatis an inhibitor of tTGase or in which a dipeptide selected from thegroup consisting of QP and LP is replaced by a constrained dipeptidemimetic compound. Such compounds are analogs of a sequence contained ingluten oligopeptides that are resistant to digestion and are believed tostimulate the autoimmune reaction that characterizes Celiac Sprue.

In another aspect, the invention provides pharmaceutical formulationscomprising a tTGase inhibitor and a pharmaceutically acceptable carrier.In one embodiment, such formulations comprise an enteric coating thatallows delivery of the active agent to the intestine, and the agents arestabilized to resist digestion or acid-catalyzed modification in acidicstomach conditions. In another embodiment, the formulation alsocomprises one or more glutenases, as described in U.S. ProvisionalApplication 60/392,782 filed Jun, 28, 2002; and U.S. ProvisionalApplication 60/428,033, filed Nov. 20, 2002, both of which areincorporated herein by reference. The invention also provides methodsfor the administration of enteric formulations of one or more tTGaseinhibitors to treat Celiac Sprue.

In another aspect, the invention provides methods for screeningcandidate compounds to determine their suitability for use in thesubject methods, by assessing the ability of a candidate agent for itsability to bind to, and/or to inhibit the activity of, tTGase. Candidateagents may also be screened for anti-allergic and anti-inflammatoryactivity by assessing their ability to bind to, and/or to inhibit theactivity of, tTGase.

In another aspect, the tTGase inhibitors and/or pharmaceuticalformulations of the present invention are useful in treating disorderswhere TGases are a factor in the disease etiology, where such disordersmay include cancer, neurological disorders, wound healing, etc. Theseconditions include Alzheimer's and Huntington's diseases, where theTGases appear to be a factor in the formation of inappropriateproteinaceous aggregates that may be cytotoxic. In diseases such asprogressive supranuclear palsy, Huntington's, Alzheimer's andParkinson's diseases, the aberrant activation of TGases may be caused byoxidative stress and inflammation.

These and other aspects and embodiments of the invention and methods formaking and using the invention are described in more detail in thedescription of the drawings and the invention, the examples, the claims,and the drawings that follow.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Celiac Sprue and/or dermatitis herpetiformis are treated by inhibitionof tissue transglutaminase. Therapeutic benefit can be enhanced in someindividuals by increasing the digestion of gluten oligopeptides, whetherby pretreatment of foodstuffs to be ingested or by administration of anenzyme capable of digesting the gluten oligopeptides, together withadministration of the tTGase inhibitor. Gluten oligopeptides are highlyresistant to cleavage by gastric and pancreatic peptidases such aspepsin, trypsin, chymotrypsin, and the like, and their prolongedpresence in the digestive tract can induce an autoimmune responsemediated by tTGase. The antigenicity of gluten oligopeptides and the illeffects caused by an immune response thereto can be decreased byinhibition of tissue transglutaminase. In another embodiment of theinvention, by also providing a means for digestion of glutenoligopeptides with glutenase, gluten oligopeptides are cleaved intofragments, thereby contributing to the prevention of the disease-causingtoxicity.

Methods and compositions are provided for the administration of one ormore tTGase inhibitors to a patient suffering from Celiac Sprue and/ordermatitis herpetiformis. In some embodiments and for some individuals,the methods of the invention remove the requirement that abstention fromingestion of glutens be maintained to keep the disease in remission. Thecompositions of the invention include formulations of tTGase inhibitorsthat comprise an enteric coating that allows delivery of the agents tothe intestine in an active form; the agents are stabilized to resistdigestion or alternative chemical transformations in acidic stomachconditions. In another embodiment, food is pretreated or combined withglutenase, or a glutenase is co-administered (whether in time or in aformulation of the invention) with a tTGase inhibitor of the invention.

The subject methods are useful for both prophylactic and therapeuticpurposes. Thus, as used herein, the term “treating” is used to refer toboth prevention of disease, and treatment of a pre-existing condition.The treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient, is a particularly important benefit provided bythe present invention. Such treatment is desirably performed prior toloss of function in the affected tissues; consequently, the prophylactictherapeutic benefits provided by the invention are also important.Evidence of therapeutic effect may be any diminution in the severity ofdisease, particularly diminution of the severity of such symptoms asfatigue, chronic diarrhea, malabsorption of nutrients, weight loss,abdominal distension, and anemia. Other disease indicia include thepresence of antibodies specific for glutens, antibodies specific fortissue transglutaminase, the presence of pro-inflammatory T cells andcytokines, and degradation of the villus structure of the smallintestine. Application of the methods and compositions of the inventioncan result in the improvement of any and all of these disease indicia ofCeliac Sprue.

Patients that can benefit from the present invention include both adultsand children. Children in particular benefit from prophylactictreatment, as prevention of early exposure to toxic gluten peptides canprevent development of the disease into its more severe forms. Childrensuitable for prophylaxis in accordance with the methods of the inventioncan be identified by genetic testing for predisposition, e.g. by HLAtyping; by family history, and by other methods known in the arL As isknown in the art for other medications, and in accordance with theteachings herein, dosages of the tTGase inhibitors of the invention canbe adjusted for pediatric use.

Because most proteases and peptidases are unable to hydrolyze the amidebonds of proline residues, the abundance of proline residues in gliadinsand related proteins from wheat, rye and barley can constitute a majordigestive obstacle for the enzymes involved. This leads to an increasedconcentration of relatively stable gluten derived oligopeptides in thegut. These stable gluten derived oligopeptides, called “toxicoligopeptides” herein, interact with tTGase to stimulate an immuneresponse that results in the autoimmune disease aspects of Celiac Sprue.

Such toxic oligopeptides include the peptide sequence PQPQLPY and longerpeptides containing that sequence or multiple copies of that sequence.This peptide sequence is a high affinity substrate for the enzyme tissuetransglutaminase (tTGase), an enzyme found on the extracellular surfacein many organs including the intestine. The tTGase enzyme catalyzes theformation of isopeptide bonds between glutamine and lysine residues ofdifferent polypeptides, leading to protein-protein crosslinks in theextracellular matrix. The tTGase enzyme acts on the peptide sequencePQPQLPY to deamidate the second Q residue, forming the peptide sequencePQPELPY. The tTGase enzyme is the primary focus of the autoantibodyresponse in Celiac Sprue. Gliadins, secalins and hordeins containseveral of the PQPQLPY sequences or sequences similar thereto rich inPro-Gin residues that are high-affinity substrates for tTGase. ThetTGase catalyzed deamidation of such sequences dramatically increasestheir affinity for HLA-DQ2, the class II MHC allele present in >90%Celiac Sprue patients. Presentation of these deamidated sequences by DQ2positive antigen presenting cells effectively stimulates proliferationof gliadin-specific T cells from intestinal biopsies of most CeliacSprue patients, providing evidence for the proposed mechanism of diseaseprogression in Celiac Sprue.

There are a number of known tTGase inhibitors that can be used in themethods of the invention. While known, these compounds have never beforebeen used to treat Celiac Sprue effectively, because the compounds havenot been administered to Celiac Sprue patients in the formulations anddosages required to deliver the active inhibitor to the small intestinein efficacious amounts. Known tTGase inhibitors include certainglutamine mimetic compounds, including compounds selected from the groupconsisting of vinylogous amides, sulfonamides, diazoketones,3-halo-4,5-dihydroisoxazoles, and 1,2,4thiadiazoles. While the presentinvention is not to be bound by a mechanistic-theory, it is believedthat these compounds provide an effective therapy-for Celiac Sprue byreversibly or irreversibly inhibiting the tTGase in the small intestine,thereby preventing it from acting on the oligopeptides comprising thePQPQLPY sequence.

PQPQLPY is a high affinity substrate for tTGase, because it has astructure that is highly complementary to the structure of the activesite of the tTGase enzyme. In particular, the peptide bonds precedingPro residues adopt trans configurations, thereby allowing the peptide toadopt an extended polyproline II helical structure. This polyproline IIhelical character is a general property of immunogenic gliadin peptides,and is an important determinant of their high affinity toward tTGase.Therefore, it has been exploited in the design of certain tTGaseinhibitors of the invention. By administering compounds that bind to theactive site of the tTGase enzyme and prevent either the binding ofimmunogenic gliadin peptides such as the 33-merLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, or their conversion toregioselectively deamidated products, a therapeutic benefit can beachieved in Celiac Sprue patients. In part, the present invention arisesout of the discoveries that the dipeptides QP and LP play an importantrole in forming the structure that binds to the active site of thetTGase enzyme and that compounds that mimic the configurations of thesedipeptides in a polyproline helix (i.e. where the imide bond adopts atrans configuration) can be used to inhibit tTGase and treat CeliacSprue. Thus, in addition to the methods for administering the glutaminemimetic compounds described above, the present invention providesmethods in which a small organic molecule that is a constrained mimeticof a dipeptide selected from the group consisting of PQ, QP, PE, PY, andLP is administered to a Celiac Sprue patient to treat celiac disease.

The tTGase inhibitors of the present invention that have structures thatmimic the conformation of the key dipeptide moieties of the tTGasesubstrate can be thought of as “tTGase inhibitory motif” or “tTGaseinhibitory moiety”. Human tTGase has a strong preference for peptidesubstrates with Type II polyproline character. This conformationalpreference is exploited by the selective tTGase inhibitors of theinvention. Dipeptide moieties of interest have the formula XP, wherein Xcan be any amino acid but is preferably selected from the groupconsisting of Q, Y, L, E, or F. Inhibitors of the invention containingsuch moieties are referred to as “peptide mimetics” or“peptidomimetics”.

Examples of dipeptidomimetics based on the trans-PQPQLPY peptide areshown below.

trans-PQPQLPY (all X-P bonds in trans configuration)

Similar dipeptidomimetics can be identified based on sequences of otherhigh-affinity gliadin peptide substrates of tTGase. Common constraineddipeptide mimetics useful for purposes of the invention also includequinozilidinone, pyrroloazepinone, indolizidinone, alkylbranchedazabicyclo[X.Y.O]alkane amino acids (Gosselin et al., J. Org. Chem.2000, 65, 2163-71; Polyak et al., J. Org. Chem. 2001, 66, 1171-80),6,5-fused bicyclic lactam (Mueller et al., Tetrahedron Lett. 1994,4091-2; Dumas, Tetrahedron Lett.1994, 1493-6, and Kim, 1997, J. Org.Chem. 62, 2847-52 ), and lactam methylene linker.

The dipeptide mimetic tTGase inhibitor compounds, like the glutaminemimetic tTGase inhibitor compounds, are believed to provide atherapeutic benefit to Celiac Sprue patients by preventing tTGase frombinding the toxic oligopeptide comprising the PQPQLPY sequence andconverting it to the PQPELPY sequence, thus preventing the initiation ofthe autoimmune response responsible for the symptoms of the disease.Alternatively, these dipeptidomimetics can be incorporated into aPQPQLPY sequence or longer peptide or peptidomimetic containing thatsequence in place of the corresponding dipeptide moiety. It is wellunderstood in the pharmaceutical arts that the more selective a drug forits intended target, and the greater affinity of a drug for its intendedtarget, the more useful the drug for the treatment of the diseaserelating to that target. Thus, while the glutamine and dipeptide mimeticinhibitors of the invention can be used to treat Celiac Sprue, therewill in some instances be a need for or benefit from compounds withgreater specificity for and affinity to tTGase. The present inventionprovides such compounds..

Thus, while beneficial therapeutic effect can be achieved by delivery ofany tTGase inhibitor to the small intestine of a Celiac Sprue patient,in a preferred embodiment, the tTGase inhibitor is contained in amolecule that is a high affinity peptide or peptidomimetic substrate oftTGase or a peptidomimetic thereof. Thus, the inhibitors of tTGaseprovided by the present invention include modified high affinity peptidesubstrates for tTGase, where one or more glutamine residues of thepeptide substrate are substituted with tTGase inhibitory moieties or oneor more dipeptides in the substrate are substituted with a dipeptidemimetic or both. In either event, the peptide or peptidomimetic does notinduce an autoimmune response in the Celiac Sprue patient.

High affinity peptide substrates for tTGase include the followingpeptides, and, with respect to the larger peptides shown, fragmentsthereof: PQPQLPY, PQPQLPYPQPQLP; LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF;QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ (from α1- and α6-gliadins);QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein);QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ (from γ-gliadin); VQWPQQQPVPQPHQPF (fromγ-gliadin), VQGQGIIQPQQPAQ (from γ-gliadin), FLQPQQPFPQQPQQPYPQQPQQPFPQ(from γ-gliadin), FSQPQQQFPQPQQPQQSFPQQQPP (from γ-gliadin), andQPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ. These peptides are resistant towardendo- and exo-proteolysis by gastric, pancreatic and small intestinalenzymes. Conservative amino acid substitutions, such as Y ->F, Q ->N/E,or L ->M, are also tolerated. Therefore, in accordance with the presentinvention, selective inhibitors of tTGase are provided by substitutingeither a glutamine that is deamidated by tTGase or a dipeptide containedin the peptide that binds in the active site of tTGase with a mimeticsuch that the resulting compound is an inhibitor of tTGase that does notstimulate the autoimmune response in a Celiac Sprue patient.

The reactive glutamines in the above proteolytically stable peptidesinclude those glutamines identified as “(Q->E)”, E being the amino acidformed by deamidation of glutamine, in the following sequences:PQP(Q->E)LPY, PQP(Q->E)LPYPQPQLP;LQLQPFPQP(Q->E)LPYPQPQLPYPQP(Q->E)LPYPQPQPF,FSQP(Q->E)Q(Q->E)FPQPQQPQQSFP(Q->E)Q(Q->E)PP, VQGQGIIQP(Q->E)QPAQ, andFLQPQQPFP(Q->E)QP(Q->E)QPYPQQPQQPFPQ. Reactive glutamine residues inother peptides can be identified by standard HPLC-MS-MS procedures, andcan be replaced by glutamine mimetics. The (Q->E) residues can bereplaced by glutamine mimetics and/or the QP and LP dipeptides in thesesequences can be replaced by dipeptidomimetics as discussed above. Thenovel tTGase inhibitors of the invention are peptides or peptidomimeticcompounds in which either a reactive glutamine or a dipeptide that bindsin the active site of tTGase or both has been replaced by a smallmolecule mimetic are referred to herein as “substituted peptides”. Inone embodiment, the tTGase inhibitors useful in the methods andcompositions of the present invention are those for which the affinityof the inhibitory moiety for the tTG active site increases (as measuredby a decrease in K_(I) or an increase in k_(inh)/K_(I)) when presentedin the context of a high affinity, proteolytically stable peptidesubstrate of the enzyme. This aspect of the invention is illustrated inthe Examples below.

Such compounds of the invention are illustrated below by compounds inwhich a reactive glutamine is replaced by a tTGase inhibitory moiety.Various tTGase inhibitory moieties useful in the methods of theinvention and that are incorporated into the novel substituted peptideand peptidomimetic tTGase inhibitors of the invention include thefollowing compounds, which are shown with variable (designated R) groupsto indicate that the compounds can be used directly as small moleculeinhibitors or incorporated into a larger dipeptide mimetic or peptide orpeptidomimetic tTGase inhibitory compound of the invention.

In the compounds shown above, R1, R2 and R3 are independently selectedfrom H, alkyl, alkenyl, cycloalkyl, aryl, heteroalkyl, heteroaryl,alkoxy, alkylthio, arakyl, aralkenyl, halo, haloalkyl, haloalkoxy,heterocyclyl, and heterocyclylalkyl groups. R1 and R2 can also be anamino acid, a peptide, a peptidomimetic, or a peptidic protectinggroups. Illustrative functional groups include: R₁ is selected from thegroup consisting of Cbz, Fmoc, Boc, PQP, Ac-PQP, PQPQLPYPQP,Ac-PQPQLPFPQP, QLQPFPQP, LQLQPFPQPLPYPQP, X₂₋₁₅-P (where X₂₋₁₅ is apeptide consisting of any 2-15 amino acid residues followed by aN-terminal proline); and R₂ is selected from the group consisting ofOMe, OtBu, Gly, Gly-NH₂, LPY, LPF-NH₂, LPYPQPQLPY, LPFPQPQLPF-NH₂,LPYPQPQLP, LPYPQPQLPYPQPQPF, LP-X₂₋₁₅ (where X₂₋₁₅ is a peptideconsisting of any 2-15 amino acid residues followed by a C-terminalproline).

Given the high selectivity of human tTGase for the peptideAc-PQPQLPF-NH₂, and

the intrinsic resistance of this peptide toward gastrointestinalproteolysis, the following tTGase inhibitors are provided by the presentinvention.

In each case, an inhibitor of the invention with greater specificity isprovided by individual or combinatorial substitution of Q, L and F withalternative amino acids. In the case of sulfonamide inhibitors, thefollowing analogs are also provided, where R is selected from an alkyl,alkenyl, cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio,arakyl, aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, orheterocyclylalkyl group. Of particular interest are the sulfonylhydrazides (R=NHR′) where R′ is H. alkyl, alkenyl, cycloalkyl, aryl,heteroalkyl, heteroaryl, alkoxy., alkylthio, arakyl, aralkenyl, halo,haloalkyl, haloalkoxy, heterocyclyl, or heterocyclylalkyl group.

In one preferred embodiment, R is a functional group whose correspondingamine is a preferred nucleophilic co-substrate of human tTGase. Forexample, the biological amine histamine is an excellent co-substrate oftTGase (kcat=20 min⁻¹, KM=40 μM). Consequently, the following compoundis a preferred tTGase inhibitor of this invention:

The synthesis of such compounds of the invention can be carried outusing methods known in the art for other purposes and the teachingsherein. For example, the synthesis of vinylogous amides such as 1 (seethe numbered structure shown below) containing an acrylamide functionhave been reported by Macedo et al. (Bioorg. Med. Chem. (2002) 10,355-360). Their ability to inhibit guinea pig tTG has been demonstrated(Marrano et al., Bioorg. Med. Chem. (2001) 9, 3231-3241). Illustrativevinylogous amide compounds of the invention include compounds in which aglutamine mimetic with an acrylamide motif such as 2 (see the numberedstructure below) is contained in a peptide or peptidomimetic having thefollowing structures: R₁ is selected from the group consisting of PQP,Ac-PQP, PQPQLPYPQP, Ac-PQPQLPFPQP, QPFPQP, LQLQPFPQPLPYPQP, or an aminoacid protecting group, including but not limited to Boc and Fmoc; and R₂is selected from the group consisting of LPY, LPF-NH₂, LPYPQPQLPY,LPFPQPQLPF-NH₂, LPYPQPQ, LPYPQPQLP, LPYPQPQLPYPQPQPF, or an amino acidprotecting group, including but not limited to OtBu, OFm or additionallyOBn or OMe.

The acrylamides can be incorporated into a high affinity peptide of theinvention by fragment condensation as illustrated below in a syntheticmethod of the invention using intermediate compounds of the invention.

a) Boc₂O, RT, 4 h, Na₂CO₃/dioxane, 95% b) C₆H₅l(OCOCF₃)₂, pyridine,DMF/H₂O, RT, 3 h, 80% c) acryl chloride, MeOH/TEA, 0° C.-RT, 12 h d)EDC, TEA, DCM e) LPF-NH₂, RT, 12 h f) HCl (gaseous), DCM, RT, 4 h g)Ac-PQP, RT, 12 h.

The tTGase inhibitory compounds of the invention from the sulfonamides,diazoketones, 1,2,4 thiadiazoles, and isoxazoles can likewise be readilyprepared using methods known in the art for other purposes and theteachings herein. To illustrate the invention with respect to suchclasses of compounds, the following amino acid analogs are employed:4-sufonamido-2-amino-butyric acid (Sab), 6-diazo-5-oxo-norleucine (Don),and acivicin (Aci),. These compounds are useful tTGase inhibitorswithout further modification, and novel tTGase inhibitors of theinvention comprise the structures of these compounds as part of alarger, high affinity inhibitor of tTGase, as illustrated by thestructures above.

Any high affinity tTGase substrate can be used to provide the scaffoldfor presenting a tTGase inhibitor moiety. Moreover, compounds not knownto be tTGase substrates can be identified by screening peptidelibraries, for example on chips or beads or displayed on phages usingreporter groups such as dansyl- or biotinyl-cadaverine, using proceduresknown in the art. Additionally, the tTGase inhibitors of the inventioncan include other moieties. As one example, in some embodiments, thetTGase inhibitor further comprise one or more proline residues C- and/orN-terminally of the glutamine mimetic-containing peptides to blockexoproteolytic degradation.

To illustrate various tTGase inhibitors of the invention, a variety ofrelatively small and large inhibitors were synthesized and tested forinhibitory activity. As examples of small molecule inhibitors, Z-Don-OMeand Z-Sab-Gly-OH were synthesized. As examples of larger inhibitors, thecompounds Ac-PQP-X-LPF-NH2, where X was Sab, a diazoketone, or acivicin,were synthesized.

Thus, Z-Don-OMe was synthesized as described (Allevi & Anatasia,Tetrahedron Asymmetry (2000) 11, 3151-3160; Pettit & Nelson, Can. J.Chem. (1986) 64, 2097-2102; Bailey & Bryans, Tetrahedron Lett. (1988)29, 2231-2234). For the synthesis of Z-Sab-Gly-OH 33, commerciallyavailable racemic homocysteine thiolactone 24 was first protected togive 25 and subsequently saponified and acetylated in situ to give thefree racemic acid 26 in high yield. Its coupling with the glycine benzylester 30 provided the dipeptide 31. Then, the conversion to thesulfonamide 32 was achieved via chlorination of the thioacetate moietyto a sulfonamide intermediate, followed by treatment with ammonia inCHCl₃. Finally, the benzyl ester protecting group was removed bysaponification with an aqueous NaOH solution.

The sulfonamide building block (Sab) 9 was incorporated into theAc-PQP-X-LPF-NH₂ scaffold by fragment condensation as illustrated in thefollowing scheme:

The diazo-ketone 10a motif was introduced into the same scaffold bypost-synthetic modification of Ac-PQP-Glu-LPF-NH₂ 40 to yield compound41.

Incorporation of the acivicin moiety 12 into the high affinity PQPXLPYscaffold was achieved by Fmoc-protection of commercially availableacivicin and Fmoc-compatible solid phase peptide chemistry as outlinedbelow.

Synthesis of peptides containing 1,2,4 thiadiazoles is described byMarrano et al., Bioorg. Med. Chem. 9, 3231-3241 (2001). Because thecarboxyl group of acivicin is not needed for tTG inhibition (Killackeyet al., Mol. Pharmacol. (1989) 35, 701-706), the3-chloro-4,5-dihydro-5-amino-isoxazole (Cai) group 13 was synthesized asdescribed (Castelhano et al., Bioorg. Chem. (1988) 16, 335-340) andcoupled C-terminally to a high-affinity peptide as depicted below:

The illustrative compounds of the invention described above were testedin a tTGase assay with recombinant human tissue transglutaminase, whichwas expressed, purified and assayed as described (Piper et al.,Biochemistry (2001) 41, 386-393). Competitive inhibition with respect tothe Cbz-Gln-Gly substrate was observed for all substrates; in all casesexcept for the Sab derivatives, irreversible inactivation of the enzymewas also observed. Importantly, all glutamine mimetics described aboveshowed significant improved specificity within a tTG-specific peptidecontext. The results also demonstrated that, while the small moleculeinhibitors can be used to inhibit tTGase, the larger compounds thatpresent the glutamine mimetic tTGase inhibitor in the context of apeptide based on the PQPQLPY sequence tended to be better inhibitors.

Thus, the present invention provides a variety of different classes ofknown and novel tTGase inhibitors. To facilitate an appreciation of theinvention, the tTGase inhibitors of the invention have in part beendescribed above with structures containing variable “R” groups that aredefined by reference to the various organic moieties that can be presentat the indicated position in the structure. Below, brief definitions areprovided for the phrases used to define the organic moieties listed foreach R group.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain radical consisting solely of carbon and hydrogen atoms, containingno unsaturation, having from one to eight carbon atoms, and which isattached to the rest of the molecule by a single bond, e.g., methyl,ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), and the like. Unless stated otherwisespecifically in the specification, the alkyl radical may be optionallysubstituted by hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro,mercapto, alkylthio, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸where each R⁸ is independently hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkylalkyl, aralkyl or aryl. Unless stated otherwise specificallyin the specification, it is understood that for radicals, as definedbelow, that contain a substituted alkyl group that the substitution canoccur on any carbon of the alkyl group.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above, e.g., methoxy, ethoxy, n-propoxy,1-methylethoxy (isopropoxy), n-butoxy, n-pentoxy, 1,1-dimethylethoxy(t-butoxy), and the like. Unless stated otherwise specifically in thespecification, it is understood that for radicals, as defined below,that contain a substituted alkoxy group that the substitution can occuron any carbon of the alkoxy group. The alkyl radical in the alkoxyradical may be optionally substituted as described above.

“Alkylthio” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above, e.g., methylthio, ethylthio,n-propylthio, 1-methylethylthio (iso-propylthio), n-butylthio,n-pentylthio, 1,1-dimethylethylthio (t-butylthio), and the like. Unlessstated otherwise specifically in the specification, it is understoodthat for radicals, as defined below, that contain a substitutedalkylthio group that the substitution can occur on any carbon of thealkylthio group. The alkyl radical in the alkylthio radical may beoptionally substituted as described above.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing at least onedouble bond, having from two to eight carbon atoms, and which isattached to the rest of the molecule by a single bond or a double bond,e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl,and the like. Unless stated otherwise specifically in the specification,the alkenyl radical may be optionally substituted by hydroxy, alkoxy,haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, —N(R⁸)₂,—C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)—C(O)-R⁸ where each R⁸ is independentlyhydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl.Unless stated otherwise specifically in the specification, it isunderstood that for radicals, as defined below, that contain asubstituted alkenyl group that the substitution can occur on any carbonof the alkenyl group.

“Aryl” refers to a phenyl or naphthyl radical. Unless stated otherwisespecifically in the specification, the term “aryl” or the prefix “ar-”(such as in “aralkyl”) is meant to include aryl radicals optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro,mercapto, alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or—N(R⁸)C(O)R⁸ where each R⁸ is independently hydrogen, alkyl, alkenyl,cycloalkyl, cycloalkylalkyl, aralkyl or aryl.

“Aralkyl” refers to a radical of the formula -R_(a)R_(b) where R_(a) isan alkyl radical as defined above and R_(b) is one or more aryl radicalsas defined above, e.g., benzyl, diphenylmethyl and the like. The arylradical(s) may be optionally substituted as described above.

“Aralkenyl” refers to a radical of the formula -R_(c)R_(b) where R_(c)is an alkenyl radical as defined above and R_(b) is one or more arylradicals as defined above, e.g., 3-phenylprop-1-enyl, and the like. Thearyl radical(s) and the alkenyl radical may be optionally substituted asdescribed above.

“Alkylene chain” refers to a straight or branched divalent hydrocarbonchain consisting solely of carbon and hydrogen, containing nounsaturation and having from one to eight carbon atoms, e.g., methylene,ethylene, propylene, n-butylene, and the like. The alkylene chain may beoptionally substituted by one or more substituents selected from thegroup consisting of aryl, halo, hydroxy, alkoxy, haloalkoxy, cyano,nitro, mercapto, alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂or —N(R⁸)C(O)R⁸ where each R⁸ is independently hydrogen, alkyl, alkenyl,cycloalkyl, cycloalkylalkyl, aralkyl or aryl. The alkylene chain may beattached to the rest of the molecule through any two carbons within thechain.

“Alkenylene chain” refers to a straight or branched divalent hydrocarbonchain consisting solely of carbon and hydrogen, containing at least onedouble bond and having from two to eight carbon atoms, e.g., ethenylene,prop-1-enylene, but-1-enylene, pent-1-enylene, hexa-1,4-dienylene, andthe like. The alkenylene chain may be optionally substituted by one ormore substituents selected from the group consisting of aryl, halo,hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio,cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸ where each R⁸is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl,aralkyl or aryl. The alkenylene chain may be attached to the rest of themolecule through any two carbons within the chain.

“Cycloalkyl” refers to a stable monovalent monocyclic or bicyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,having from three to ten carbon atoms, and which is saturated andattached to the rest of the molecule by a single bond, e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decalinyl and thelike. Unless otherwise stated specifically in the specification, theterm “cycloalkyl” is meant to include cycloalkyl radicals which areoptionally substituted by one or more substituents independentlyselected from the group consisting of alkyl, aryl, aralkyl, halo,haloalkyl, hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto,alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸where each R⁸ is independently hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkylalkyl, aralkyl or aryl.

“Cycloalkylalkyl” refers to a radical of the formula -R_(a)R_(d) whereR_(a) is an alkyl radical as defined above and R_(d) is a cycloalkylradical as defined above. The alkyl radical and the cycloalkyl radicalmay be optionally substituted as defined above.

“Halo” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like.

“Haloalkoxy” refers to a radical of the formula —OR_(c) where R_(c) isan haloalkyl radical as defined above, e.g., trifluoromethoxy,difluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy,1-fluoromethyl-2-fluoroethoxy, 3-bromo-2-fluoropropoxy,1-bromomethyl-2-bromoethoxy, and the like.

“Heterocyclyl” refers to a stable 3- to 15-membered ring radical whichconsists of carbon atoms and from one to five heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur. For purposes ofthis invention, the heterocyclyl radical may be a monocyclic, bicyclicor tricyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical may be optionally oxidized; the nitrogen atom may beoptionally-quatemized; and the heterocyclyl radical may be aromatic orpartially or fully saturated. The heterocyclyl radical may not beattached to the rest of the molecule at any heteroatom atom. Examples ofsuch heterocyclyl radicals include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzothiadiazolyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, carbazolyl, cinnolinyl,decahydroisoquinolyl, dioxolanyl, furanyl, furanonyl, isothiazolyl,imidazolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, indolyl,indazolyl, isoindolyl, indolinyl, isoindolinyl, indolizinyl, isoxazolyl,isoxazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolyl,oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl,quinolinyl, quinuclidinyl, isoquinolinyl, thiazolyl, thiazolidinyl,thiadiazolyl, triazolyl, tetrazolyl, tetrahydrofuryl, triazinyl,tetrahydropyranyl, thienyl, thiamorpholinyl, thiamorpholinyl sulfoxide,and thiamorpholinyl sulfone. Unless stated otherwise specifically in thespecification, the term “heterocyclyl” is meant to include heterocyclylradicals as defined above which are optionally substituted by one ormore substituents selected from the group consisting of alkyl, halo,nitro, cyano, haloalkyl, haloalkoxy, aryl, heterocyclyl,heterocyclylalkyl, —OR⁸, -R⁷—OR⁸, —C(O)OR⁸, -R⁷—C(O)OR⁸, —C(O)N(R⁸)₂,—N(R⁸)₂, -R⁷—N(R⁸)₂, and —N(R⁸)C(O)R⁸ wherein each R⁷ is a straight orbranched alkylene or alkenylene chain and each R⁸ is independentlyhydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl.

“Heterocyclylalkyl” refers to a radical of the formula -R_(a)R_(e) whereR_(a) is an alkyl radical as defined above and R_(e) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. The heterocyclyl radical may beoptionally substituted as defined above.

In the formulas provided herein, molecular variations are included,which may be based on isosteric replacement. “lsosteric replacement”refers to the concept of modifying chemicals through the replacement ofsingle atoms or entire functional groups with alternatives that havesimilar size, shape and electro-magnetic properties, e.g. O is theisosteric replacement of S, N, COOH is the isosteric replacement oftetrazole, F is the isosteric replacement of H, sulfonate is theisosteric replacement of phosphate etc.

As used herein, compounds which are “commercially available” may beobtained from standard commercial sources including Acros Organics(Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wiss., including SigmaChemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H., Maybridge Chemical Co. Ltd. (CornwallU.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc. (WaterburyConn.), Polyorganix (Houston Tex.), Pierce Chemical Co. (Rockford Ill.),Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc.(New Brunswick, N.J.), TCI America (Portland Oreg.), Trans WorldChemicals, Inc. (Rockville Md.), Wako Chemicals USA, Inc. (RichmondVa.), Novabiochem and Argonaut Technology.

As used herein, “suitable conditions” for carrying out a synthetic stepare explicitly provided herein or may be discerned by reference topublications directed to methods used in synthetic organic chemistry.The reference books and treatise set forth above that detail thesynthesis of reactants useful in the preparation of compounds of thepresent invention, will also provide suitable conditions for carryingout a synthetic step according to the present invention.

As used herein, “methods known to one of ordinary skill in the art” maybe identified though various reference books and databases. Suitablereference books and treatise that detail the synthesis of reactantsuseful in the preparation of compounds of the present invention, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., NewYork; S. R. Sandier et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modem SyntheticReactions”, 2nd Ed., W. A Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Specificand analogous reactants may also be identified through the indices ofknown chemicals prepared by the Chemical Abstract Service of theAmerican Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., www.acs.org may be contacted formore details). Chemicals that are known but not commercially availablein catalogs may be prepared by custom chemical synthesis houses, wheremany of the standard chemical supply houses. (e.g., those listed above)provide custom synthesis services.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline and caffeine.

The tTGase inhibitors, or their pharmaceutically acceptable salts maycontain one or more asymmetric centers and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L) for amino acids. The present invention is meant to includeall such possible isomers, as well as, their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, such as reverse phase HPLC. Whenthe compounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included.

The present invention provides the tTGase inhibitors in a variety offormulations for therapeutic administration. In one aspect, the agentsare formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and areformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the tTGase inhibitors is achievedin various ways, although oral administration is a preferred route ofadministration. In some formulations, the tTGase inhibitors are systemicafter administration; in others, the inhibitor is localized by virtue ofthe formulation, such as the use of an implant that acts to retain theactive dose at the site of implantation.

In some pharmaceutical dosage forms, the tTGase inhibitors areadministered in the form of their pharmaceutically acceptable salts. Insome dosage forms, the tTGase inhibitor is used alone, while in others,the tTGase is used in combination with another pharmaceutically activecompounds. In the latter embodiment, the other active compound is, insome embodiments, a glutenase that can cleave or otherwise degrade atoxic gluten oligopeptide, as described in the Examples below. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, the agents are used alone or in combination withappropriate additives to make tablets, powders, granules or capsules,for example, with conventional additives, such as lactose, mannitol,corn starch or potato starch; with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and in some embodiments, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations compriseenteric coatings, so that the active agent is delivered to theintestinal tract. Enteric formulations are often used to protect anactive ingredient from the strongly acid contents of the stomach. Suchformulations are created by coating a solid dosage form with a film of apolymer that is insoluble in acid environments and soluble in basicenvironments. Exemplary films are cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methylcellulose phthalateand-hydroxypropyl methylcellulose acetate succinate, methacrylatecopolymers and cellulose acetate phthalate.

Other enteric formulations of the tTGase inhibitors of the inventioncomprise engineered polymer microspheres made of biologically erodablepolymers, which display strong adhesive interactions withgastrointestinal mucus and cellular linings, can traverse both themucosal absorptive epithelium and the follicle-associated epitheliumcovering the lymphoid tissue of Peyer's patches. The polymers maintaincontact with intestinal epithelium for extended periods of time andactually penetrate it, through and between cells. See, for example,Mathiowitz et al. (1997) Nature 386 (6623): 410-414. Drug deliverysystems can also utilize a core of superporous hydrogels (SPH) and SPHcomposite (SPHC), as described by Dorkoosh et al. (2001) J ControlRelease 71(3):307-18.

In another embodiment, the tTGase inhibitor or formulation thereof isadmixed with food, or used to pre-treat foodstuffs containing glutens.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form,” refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of tTGase inhibitor calculated in an amount sufficient toproduce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the unitdosage forms of the present invention depend on the particular complexemployed and the effect to be achieved, and the pharmacodynamicsassociated with each complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Depending on the patient and condition being treated and on theadministration route, the tTGase inhibitor is administered in dosages of0.01 mg to 500 mg V/kg body weight per day, e.g. about 20 mg/day for anaverage person. Dosages are appropriately adjusted for pediatricformulation. Those of skill will readily appreciate that dose levels canvary as a function of the specific inhibitor, the diet of the patientand the gluten content of the diet, the severity of the symptoms, andthe susceptibility of the subject to side effects. Some of theinhibitors of the invention are more potent than others. Preferreddosages for a given inhibitor are readily determinable by those of skillin the art by a variety of means. A preferred means is to measure thephysiological potency of a given compound.

The methods of the invention are useful in the treatment of individualssuffering from Celiac Sprue and/or dermatitis herpetiformis, byadministering an effective dose of a tTGase inhibitor, through apharmaceutical formulation, and the like. Diagnosis of suitable patientsmay utilize a variety of criteria known to those of skill in the art. Aquantitative increase in antibodies-specific for gliadin, and/or tissuetransglutaminase is indicative of the disease. Family histories and thepresence of the HLA alleles HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8[DQ(a1*0301, b1*0302)] are indicative of a susceptibility to thedisease. Moreover, as tTG plays an important role in other diseases,such as Huntington's disease and skin diseases in addition to dermatitisherpetiformis, a variety of formulated versions of the compounds of theinvention (e.g. topical formulations, intravenous injections) are usefulfor the treatment of such medical conditions. These conditions includeAlzheimer's and Huntington's diseases, where the TGases appear to be afactor in the formation of inappropriate proteinaceous aggregates thatmay be cytotoxic. In diseases such as progressive supranuclear palsy,Huntington's, Alzheimer's and Parkinson's diseases, the aberrantactivation of TGases may be caused by oxidative stress and inflammation.

Therapeutic effect is measured in terms of clinical outcome, or byimmunological or biochemical tests. Suppression of the deleteriousT-cell activity can be measured by enumeration of reactive Th1 cells, byquantitating the release of cytokines at the sites of lesions, or usingother assays for the presence of autoimmune T cells known in the art.Also both the physician and patient can identify a reduction in symptomsof a disease.

Various methods for administration are employed in the practice of theinvention. In one preferred embodiment, oral administration, for examplewith meals, is employed. The dosage of the therapeutic formulation canvary widely, depending upon the nature of the disease, the frequency ofadministration, the manner of administration, the clearance of the agentfrom the patient, and the like. The initial dose can be larger, followedby smaller maintenance doses. The dose can be administered asinfrequently as weekly or biweekly, or more often fractionated intosmaller doses and administered daily, with meals, semi-weekly, and thelike, to maintain an effective dosage level.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature), but someexperimental errors and deviations may be present. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

EXAMPLE 1 Synthesis of Glutamine Mimetic tTGase Inhibitors

Synthesis of N-(Carbobenzyloxy)-D,L-homocysteine thiolactone (25). To asolution of DL-homocysteine thiolactone hydrochloride (1 eq.) in anaqueous solution of Na₂CO₃ (10 eq.) and dioxane (v/v), cooled to 0° C.,benzylchloroformate (1 eq) in dioxane is added. After 20 h at roomtemperature, the bulk of the dioxane is evaporated and the resultingaqueous solution extracted with AcOEt. The combined extracts are washedwith brine, dried over sodium sulfate and evaporated. The crude productis triturated in ether and finally filtered. White solid. Yield 95%. ¹HNMR (CDCl₃) δ1.98 (m, 1H), 2.87 (m, 1H), 3,24-3.34 (m, 2H), 4. 31 (m,1H), 5.12 (s, 2H), 7.35 (m, 5H)

Synthesis of S-acetyl-N-(carbobenzyloxy)-D,L-homocysteine (26). Asolution of N-(Carbobenzyloxy) -D,L-homocysteine thiolactone 25 (1 eq.)in THF:H₂O 1.5:0.5 was degassed three times. A solution of 6M aqueousdegassed KOH (3 eq.), was added the thiolactone solution. After thesolution was stirred at room temperature for 1.5 h, acetic anhydride(5.3 eq.) was then added dropwise with continued cooling (ice bath),maintaining a temperature of <27 ° C. After an additional 30 min. atroom temperature, the reaction was acidified with 6N aqueous HCl to pH4.3, and then concentrated in vacuo. The concentrate was acidifiedfurther with additional 6N aqueous HCl to pH 2.6. The product wasextracted with EtOAc. The combined organic extracts were washed threetimes with saturated brine, dried (Na₂SO₄), filtered, and concentratedunder vacuum to afford a tacky white solid. The residue was azeotropedthree times with toluene to remove residual acetic acid. The solid wascollected by filtration using hexane:EtOAc 1:1 and dried to affordracemic 26, free acid form, as a white solid. Yield 85%. TLC R_(f)0.48(EtOAC:AcOH 98:2). ¹H NMR (CDCl₃) δ1.99 (m, 1H), 2.08 (m, 1H), 2.29 (s,3H), 2.86-2.98 (m, 2H), 4.14 (m, 1H), 5.12 (s, 2H), 7.35 (m, 5H).

Synthesis of (31). To a solution of the free racemic acid ofS-acetyl-N-(carbobenzyloxy) -D,L-homocysteine 26 (1 eq.) in DCM at 0° C.was added 1-hydroxybenzotrizole hydrate (HOBt, 1.1 eq.), followed by1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDPI, 1eq.). The resulting suspension was stirred at 0° C. for 30 min and thena solution of glycine benzylester 30 (1 eq.) in DCM was added, followedby dropwise addition of a solution of 4-dimethylaminopyridine (DMAP, 1.2eq.) in DCM. The resulting suspension was stirred at room temperaturefor 20 h. The reaction mixture was partitioned between EtOAc and 5%aqueous NaHPO₄. The separated organic layer was then washed with 5%aqueous NaHPO₄, satured aqueous Na₂CO₃, H₂O, and brine, dried overNa₂SO₄, and filtered. Yhe filtrate was concentrated in vacuo, and theresidu was flash chromatographed on a short silica gel column to affordthe pure dipeptide 31 as a colorless oil. Yield 75%. ¹H NMR (CDCl₃)δ1.98 (m, 1H), 2.05-2.13 (m, 3H), 2.29 (s, 3H), 2.86 (m, 1H), 2.98 (m,1H), 4.14 (m, 1H), 5.10-5.14 (m, 4H), 7.28-7.42 (m, 10H).

Synthesis of (32). A solution of 31 (1 eq.) and NaOAc (10 eq.) inHOAc:H₂O 5:1 was stirred below 10° C. Gaseous chlorine was bubbled intothe solution. After 10 min argon was blown through the yellow mixturefor 10 min to remove excess Cl₂ and the solvent was evaporated. Theresidue was partitioned between EtOAc and H₂O. The EtOAc solution waswashed with brine, dried, and evaporated to the yellow oilysulfonylchloride. This product was used without further purification inthe next stage. A solution of the crude sulfonylchloride (1 eq.) inCHCl₃ was stirred below 10° C. Gaseous ammoniac was bubbled into thesolution. After 20 min, the mixture was stirred for 30 min, allowed towarm to room temperature, and evaporated to dryness. The residue waspartitioned between EtOAc and H₂O. The EtOAc solution was washed withbrine, dried, and evaporated to a colorless oil. Yield 75%. ¹H NMR(CDCl₃) δ2.01 (m, 1H), 2.13 (s, 2H), 2.22-2.32 (m, 1H), 3.21-3.31 (m,2H), 4.14 (m, 1H), 5.10-5.14 (m, 4H), 7.27-7.41 (m,₁₀H).

Synthesis of (33). The benzyl ester 32 (1 eq.) was stirred for 2 h in amixture of aqueous 1N NaOH:EtOH 1.2:3 (10 eq.). The reaction mixture wasevaporated to dryness and the residue was dissolved in a small amount ofH₂O. The solution was filtered into a centrifuge tube and acidified topH 3. The gelatinous precipitate was isolated by centrifugation, washedwith CHCl₃, and dried to a white solid. Yield 60%. MS m/z 372.3 [M-H⁻]⁻.

Synthesis of Fmoc-Acivicin 45. 3.1 ml of a 0.75 M solution ofFmoc-N-hydroxysuccinimide in acetone was added to 0.4 g acivicin (2.25mmol, Biomol) dissolved in 3.1 ml of a 10% Na₂CO₃ aqueous solution. Theslurry was stirring for 4 hours and the pH of was maintained at 9.0 byaddition of Na₂CO₃. The solvent was removed by rotary evaporation, theresidual solid was dissolved in 0.6 M HCL, extracted with ethyl acetateand concentrated to a yellow oil. Recrystallization from ethyl acetate:hexane yielded 0.62 9 (1.55 mmol, 70%) of the desired product as whitecrystals. R_(f) (CH₂Cl₂: iPrOH: AcOH=100:3:1)=0.3 ¹H (d⁶-acetone, 200MHz) cpm=7.87 ArH (2H, d, J=7.4 Hz); 7.73 ArH (2H, d, J=7 Hz); 7.28-7.48ArH (4H, m); 7.17 NH (1H, d, J=8 Hz); 5.22 CH₂CHO (1H, m); 4.66 (1H, q,J=4.4 Hz); 4.2-4.4 (3H); 3.6-3.4 (2H). m [M-Na]⁺=423.4, 425.3 g/mol.

Synthesis of Pro-Gln-Pro-Aci-Leu-Pro-Tyr 46. PQPAciLPY was synthesizedby standard Fmoc solid phase chemistry using Fmoc-acivicin andcommercially available building blocks in a 25 μmol scale. Preparativereversed phase HPLC purification yielded 4 OD₂₇₅ (3.4 μmol, 14%). LC-MS:R_(t)=12 min, [M+H]⁺=874.6.

Synthesis of Ac-Pr-Gl-Pr-DonLeu-Pr-PheNH₂41. 72 mg (8.3 μmol) ofHPLC-purified, lyophilized Ac-Pro-Gln-Pro-Glu-Leu-Pro-Phe-NH₂ in 1 mlTHF and 15 μl (135μmol) N-methyl morpholine were mixed with 13 μl(100μmol) at 0° C., followed by addition of up to 0.5 mol of a saturateddiazomethane solution in dry ether generated from Diazald as describedby the supplier. After 1 hour the solvents were evaporated, the residualsolid was extracted with ethyl ester and a 5% aqueous solution ofNH₄HCO₃, and the combined aqueous phases were concentrated by rotaryevaporation.. The crude product was purified by preparative reversedphase HPLC on a Beckman Ultrashpere C18 column (15×2.54 cm) using a 1%NH₄HCO₃ as buffer A and 0.5% NH₄HCO₃, 80% acetonitrile as buffer B. Theproduct eluting at 22.5% buffer B was concentrated yielding 16 mg (150OD₂₇₅) of lyophyllized product. [M+Na]⁺=914.4.

Synthesis of (S)-2-Benzyloxycarbonylamino-4-suffamoyl-butyric acid ethylester (a) (Cbz-homocys)₂

1.00 g (3.65 mmol) of L-homocystine (Bachem, Calif.) was dissolved in 15ml of 1:1 (v/v) mixture of 1,4-dioxane and water, and NaOH (0.30 g, 2.0eq) was added. To the solution cooled down to 0° C., benzylchloroformate (1.27 ml, 2.3 eq) was added dropwise as the pH of thesolution was maintained slightly basic by simultaneous addition of 1 NNaOH. After stirring for 1 hr, the solution was washed with ether,acidified with 6 N HCl and extracted with ethyl acetate. The organiclayer was washed with brine and dried over Na₂SO₄. After filtration, thesolvent was removed by evaporation and the residue was dried undervacuum to give the title compound as a white solid (1.83 g, 92%). ¹H NMR(DMSO-d₆, 200 MHz): δ=7.59(d, 2H, J=8.0 Hz), 7.29-7.26(m, 10H), 4.96(s,4H), 4.03-3.97(m, 2H), 2.70-2.62(m, 4H), 2.05-1.84(m, 4H) MS (ESl):m/z=536.9 [M+H]⁺, 559.1 [M+Na]⁺(b) (Cbz-homocys-OEt)₂

1.00 g (1.86 mmol) of (Cbz-homocys)₂ was dissolved in 10 ml EtOH. To thesolution cooled down to 0° C., SOCl₂ (0.33 ml, 2.4 eq) was addeddropwise and the stirring was continued overnight at room temperature.The solvent was removed by evaporation and the residue was redissolvedin ethyl acetate. The solution was washed with sat. NaHCO₃ solution andbrine, and dried over Na₂SO₄. After filtration, the solvent was removedby evaporation and the residue was dried under vacuum to give the titlecompound as a white solid (1.10 g, quant.). ¹H NMR (CDC1₃, 200 MHz):δ=7.30-7.27(m, 10H), 5.40(d, 2H, J=8.2 Hz), 5.04(s, 4H), 4.43-4.38(m,2H), 4.15(q, 4H, J=7.0 Hz), 2.69-2.61(m, 4H), 2.20-1.94(m, 4H), 1.22(t,3H, J =7.0 Hz) MS (ESl): m/z=592.9 [M+H]⁺, 615.2 [M+Na]⁺(c)(S)-2-Benzyloxycarbonylamino-4-sulfamoyl-butyric acid ethyl ester

1.00 g (1.77 mmol) of (Cbz-homocys-OEt)₂ was dissolved in 12 ml of 2:1(v/v) mixture of CCl₄ and EtOH. Cl₂ (g) was bubbled through the solutioncooled down to 0° C. for 1 hr. Stirring was continued for 20 min at roomtemperature with Ar bubbling. The solvents were removed by evaporationand the residue was dried under vacuum.

This (S)-2-benzyloxycarbonylamino4-chlorosulfonyl-butyric acid ethylester was dissolved in 10 ml CH₂Cl₂ and NH₃ (g) was bubbled through thesolution at 0° C. for 30 min. The solvent was removed by evaporation andthe residue was redissolved in ethyl acetate. The solution was washedwith brine and dried over Na₂SO₄. After filtration, the solvent wasremoved by evaporation and the residue was purified by SiO₂chromatography to give the title compound as a white solid (0.95 g,82%). ¹H NMR (CDCl₃, 200 MHz): δ=7.32-7.30(m, 5H), 5.49(d, 1H, J=8.4Hz), 5.07(s, 2H), 4.71 (br, 2H), 4.50-4.45(m, 1 H), 4.18(q, 2H, J=7.2Hz), 3.21-3.13(m, 2H), 2.42-2.14(m, 2H), 1.24(t, 3H, J=7.2 Hz) MS (ESI):m/z=367.1 [M+Na]⁺

Synthesis of (S)-2-Benzyloxycarbonylamino-4-hydrazinosulfonyl-butyrcacid ethyl ester (S)-2-benzyloxycarbonylamino-4-chlorosulfonyl-butyricacid ethyl ester, prepared from 0.10 g of (Cbz-homocys-OEt)₂ as above,was reacted with hydrazine monohydrate (38 μl, 2.2 eq) in 2 ml CH₂Cl₂for 1 hr. The solution was diluted with ethyl acetate and washed with0.1 N HCl, sat. NaHCO₃ solution and brine. The solvents were evaporatedand the residue was purified to by SiO₂ chromatography to give the titlecompound as clear oil (84 mg, 70%). ¹H NMR (CDCl₃, 200 MHz):δ=7.30-7.28(m, 5H), 5.54(d, 1H, J=8.4 Hz), 5.05(s, 2H), 4.45-4.40(m,1H), 4.16(q, 2H, J=7.0 Hz), 4.11(br, 3H), 3.24-3.08(m, 2H), 2.38-2.02(m,2H), 1.22(t, 3H, J=7.0 Hz) MS (ESl): m/z=352.1 [M+Na]⁺

Synthesis of(S)-2-Benzyloxycarbonylamino-4-phenylhydrazinosulfonyl-butyric acidethyl ester. According to the procedure described for the synthesis of(S)-2-Benzyloxycarbonylamino-4-hydrazinosulfonyl-butyric acid ethylester, the title compound was obtained from phenylhydrazine as slightlyorange oil. ¹H NMR (CDCl₃, 200 MHz): δ=7.29-7.15(m, 9H), 6.87(d, 2H,J=7.0 Hz), 6.09(s,1H), 5.31 (d, 1H, J=7.8 Hz), 5.02(s, 2H), 4.34-4.30(m,1H), 4.10(q, 2H, J=7.2 Hz), 3.07-2.99(m, 2H), 2.36-2.04(m, 2H), 1.18(t,3H, J=7.2 Hz) MS (ESl): m/z=458.0 [M+Na]⁺

Inhibition of tTG. tTG (9 μM) was inactivated in 200 mM MOPS, pH=7.1, 5mM CaCl₂, 1 mM ETDA at 30° C. containing 0-600 μMPro-Gln-Pro-Aci-Leu-Pro-Tyr. Every 20 minutes a 40 μl aliquot wasremoved and residual tTG activity was assayed in 0.5 ml reactioncontaining 200 mM MOPS, pH=7.1, 5 mM CaCl₂, 1 mM ETDA, 10 mMα-ketoglutarate, 180U/ml glutamate dehydrogenase (Biozyme laboratories)at 30° C. for 20 minutes by measuring the decrease of absorption at 340nm. Residual activity was corrected by the corresponding uninhibited tTGreaction (0 μM inhibitor) and fitted to an exponential decay. Kineticparameters were obtained by double-reciprocal plotting of the apparentsecond-order inactivation constant or, for sulfonamides and sulfonylhydrazides, by fitting the data for reversible inhibitors to a standardMichaelis Menten equation with a competitive inhibition constant. Theresults of these inhibition experiments are shown in Tables 1, and 2 and3 below. TABLE 1 Kinetic parameters of catalysis and inhibition oftissue transglutaminase by reactive glutamine peptide analogs. Thereactive glutamine (—X—) in the peptide substrate was substituted by theinhibitory residue acivicin (Aci) or 6-diazo-5-oxo-norleucine (DON).Reactive Gln Aci DON Motif: k_(cat) K_(M) k_(cat)/K_(M) k_(inh) K_(I)k_(inh)/K_(I) k_(inh) K_(I) k_(inh)/K_(I) Scaffold: [min⁻¹] [M][min⁻¹M⁻¹] [min⁻¹] [M] [min⁻¹M⁻¹] [min⁻¹] [M] [min⁻¹M⁻¹] H—X—OH — >0.2≦2 0.015 0.087 0.17 0.025 0.13 0.2 Cbz-X— — >0.03  90 — — — 0.12 1.35 ×10⁻⁴ 890 OMe PQP—X— 28 3 × 10⁻⁴ 8.2 × 10⁻⁴ 0.014 7.8 × 10⁻⁴ 18 — — — LPYAc—PQP— 40 4 × 10⁻⁴ 9.7 × 10⁴  — — — 0.2   7 × 10⁻⁸ 2.9 × 10⁶ X—LPF— NH₂

TABLE 2 Kinetic parameters of catalysis and inhibition of tissuetransglutaminase by Sab and Z-Sab-Gly. Compound Sab Z-Sab-Gly K_(I)[mM] >200 8 k_(inh) [min⁻¹] — — k_(inh)/K_(I) — — [mM⁻¹min⁻¹]

TABLE 3 Tissue transglutaminase inhibition by sulfonamides and sulfonylhydrazides tested compound inhibition constant (M)(S)-2-Benzyloxycarbonylamino-4-sulfamoyl- 4.4 × 10⁻³ butyric acid ethylester (S)-2-Benzyloxycarbonylamino-4-hydra- 2.2 × 10⁻³zinosulfonyl-butyric acid ethyl ester(S)-2-Benzyloxycarbonylamino-4-phenyl- 1.3 × 10⁻⁴hydrazinosulfonyl-butyric acid ethyl ester

The above results demonstrate that the compounds tested have tTGaseinhibitory activity.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. Moreover, due to biological functionalequivalency considerations, changes can be made in protein structurewithout affecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1. A method of treating Celiac Sprue and/or dermatitis herpetiformis,the method comprising: administering to a patient an effective dose of atTGase inhibitor; wherein said tTGase inhibitor attenuates glutentoxicity in said patient.
 2. The method of claim 1, wherein said tTGaseinhibitor is or comprises a glutamine mimetic.
 3. The method of claim 1,wherein said tTGase inhibitor is administered with a glutenase.
 4. Themethod according to claim 1, wherein said tTGase inhibitor isadministered orally.
 5. The method according to claim 1, wherein saidtTGase inhibitor is contained in a formulation that comprises an entericcoating.
 6. A formulation for use in treatment of Celiac Sprue and/ordermatitis herpetiformis, comprising: an effective dose of a tTGaseinhibitor and a pharmaceutically acceptable excipient.
 7. Theformulation of claim 6, wherein said tTGase inhibitor is selected fromthe group of compounds consisting of a glutamine mimetic, a compoundcomprising a glutamine mimetic, a dipeptide mimetic of a dipeptideselected from the group consisting of QP and LP and a compoundcomprising said dipeptide mimetic.
 8. A tTGase inhibitor that is apeptidase resistant polypeptide comprising one or more tTGase inhibitorymoieties.
 9. The tTGase inhibitor of claim 8, wherein said tTGaseinhibitory moiety is a glutamine analog.
 10. The tTGase inhibitor ofclaim 9, wherein said tTGase inhibitory moiety is selected from thegroup consisting of:

wherein R1, R2 and R3 are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, arakyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups, and wherein R1 and R2 can also be an aminoacid, a peptide, a peptidomimetic, or a peptidic protecting group, andwherein R1 can additionally be selected from the group consisting ofCbz, Fmoc, Boc, PQP, Ac-PQP, PQPQLPYPQP, Ac-PQPQLPFPQP, QLQPFPQP,LQLQPFPQPLPYPQP, X₂₋₁₅-P (where X₂₋₁₅ is a peptide consisting of any2-15 amino acid residues followed by a N-terminal proline); R2 canadditionally be selected from the group consisting of OMe, OtBu, Gly,Gly-NH₂, LPY, LPF-NH₂, LPYPQPQLPY, LPFPQPQLPF-NH₂, LPYPQPQLP,LPYPQPQLPYPQPQPF, LP-X₂₋₁₅ (where X₂₋₁₅ is a peptide consisting of any2-15 amino acid residues followed by a C-terminal proline); and R3 canadditionally be a sulfonyl hydrazide (NHR′) where R′ is selected from H,alkyl, alkenyl, cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy,alkylthio, arakyl, aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl,and heterocyclylalkyl group.
 11. The tTGase inhibitor of claim 9,wherein said glutenase resistant peptide is selected from the groupconsisting of: PQPQLPY, PQPQLPYPQPQLP LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ;QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF; QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ;VQWPQQQPVPQPHQPF, VQGQGIIQPQQPA; FLQPQQPFPQQPQQPYPQQPQQPFPQ;FSQPQQQFPQPQQPQQSFPQQQPP; and QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ.


12. A tTGase inhibitor comprising: a compound that mimics a bindingactivity of tTGase reactive dipeptide having the amino acid sequence ofXP; wherein said X is any amino acid.
 13. The tTGase inhibitor of claim12, wherein X is selected from the group consisting of Q, L, Y and F.14. The method according to claim 1, wherein said tTGase inhibitorcomprises: a compound that mimics a binding activity of tTGase reactivedipeptide having the amino acid sequence of XP; wherein said X is anyamino acid.
 15. The method according to claim 14, wherein X is selectedfrom the group consisting of Q, L, Y and F.
 16. The method according toclaim 1, wherein said tTGase inhibitor has the formula selected from thegroup consisting of:

wherein R1, R2 and R3 are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, arakyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups, and wherein R1 and R2 can also be an aminoacid, a peptide, a peptidomimetic, or a peptidic protecting group, andwherein R1 can additionally be selected from the group consisting ofCbz, Fmoc, Boc, PQP, Ac-PQP, PQPQLPYPQP, Ac-PQPQLPFPQP, QLQPFPQP,LQLQPFPQPLPYPQP, X₂₋₁₅-P (where X₂₋₁₅ is a peptide consisting of any2-15 amino acid residues followed by a N-terminal proline); and R₂ canadditionally be selected from the group consisting of OMe, OtBu, Gly,Gly-NH₂, LPY, LPF-NH₂, LPYPQPQLPY, LPFPQPQLPF-NH₂, LPYPQPQLP,LPYPQPQLPYPQPQPF, LP-X₂₋₁₅ (where X₂s₁₅ is a peptide consisting of any2-15 amino acid residues followed by a C-terminal proline) ); and R3 canadditionally be a sulfonyl hydrazide (NHR′) where R′ is selected from H,alkyl, alkenyl, cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy,alkylthio, arakyl, aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl,and heterocyclylalkyl group.
 17. The formulation according to claim 6,wherein said tTGase inhibitor comprises: a compound that mimics abinding activity of tTGase reactive dipeptide having the amino acidsequence of XP; wherein said X is any amino acid.
 18. The formulationaccording to claim 17, wherein X is selected from the group consistingof Q, L, Y and F.
 19. The formulation according to claim 18, whereinsaid compound has the formula selected from the group consisting of:

wherein R1, R2 and R3 are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, aralkyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups, and wherein R1 and R2 can also be an aminoacid, a peptide, a peptidomimetic, or a peptidic protecting group, andwherein R1 can additionally be selected from the group consisting ofCbz, Fmoc, Boc, PQP, Ac-PQP, PQPQLPYPQP, Ac-PQPQLPFPQP, QLQPFPQP,LQLQPFPQPLPYPQP, X₂₋₁₅-P (where X₂₋₁₅ is a peptide consisting of any2-15 amino acid residues followed by a N-terminal proline); and R₂ canadditionally be selected from the group consisting of OMe, OtBu, Gly,Gly-NH₂, LPY, LPF-NH₂, LPYPQPQLPY, LPFPQPQLPF-NH₂, LPYPQPQLP,LPYPQPQLPYPQPQPF, LP-X₂₋₁₅ (where X₂₋₁₅ is a peptide consisting of any2-15 amino acid residues followed by a C-terminal proline) ); and R3 canadditionally be a sulfonyl hydrazide (NHR′) where R′ is selected from H,alkyl, alkenyl, cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy,alkylthio, arakyl, aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl,and heterocyclylalkyl group.
 20. A use of tissue transglutaminaseinhibitors as set forth of claim 8 for the treatment of a disorder wheretissue transglutaminase is a factor in disease etiology.
 21. The useaccording to claim 20, wherein said disorder is a neurological disorder,cancer or wound healing.